{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T14:00:04Z","timestamp":1790258404484,"version":"4.1.0"},"reference-count":20,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T00:00:00Z","timestamp":1789516800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T00:00:00Z","timestamp":1789516800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22307013"],"award-info":[{"award-number":["22307013"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22207014"],"award-info":[{"award-number":["22207014"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22277010"],"award-info":[{"award-number":["22277010"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22477013"],"award-info":[{"award-number":["22477013"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Expanding the repertoire of usable E3 ubiquitin ligases remains a critical challenge in the field of targeted protein degradation. For the emerging E3 ligase Kelch domain\u2010containing protein 2 (KLHDC2), currently reported ligands have a carboxylate moiety in common that mimics the natural degron but compromises cellular permeability. Here we report the discovery of carboxylate\u2010free ligands for KLHDC2 through high\u2010throughput screening and cellular functional evaluation. A fluorescence polarization\u2010based screening assay identified\n                    <jats:bold>NL1<\/jats:bold>\n                    as a micromolar KLHDC2 binder, which was confirmed by orthogonal biophysical methods. Although the structure\u2013activity study did not improve affinity, a degron reporter assay demonstrated measurable cellular target engagement. Encouraged by these results, an expanded screen further identified\n                    <jats:bold>NL2<\/jats:bold>\n                    as a neutral ligand with improved cellular activity. Computational analysis suggests that\n                    <jats:bold>NL2<\/jats:bold>\n                    adopts a distinct binding mode, in which the conformational flexibility of two loops of KLHDC2 enables accommodation of its aromatic scaffold, while hydrophobic and \u03c0\u2010driven interactions compensate for the canonical ionic contacts of other carboxylate\u2010containing ligands. This work demonstrates that KLHDC2 can accommodate non\u2010carboxylate scaffolds and expands the chemical space for developing KLHDC2\u2010based targeted protein degradation strategies.\n                  <\/jats:p>","DOI":"10.1002\/pro.70787","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T12:56:08Z","timestamp":1789563368000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["High\u2010throughput identification of carboxylate\u2010free ligands for the\n                    <scp>E3<\/scp>\n                    ligase Kelch domain\u2010containing protein 2"],"prefix":"10.1002","volume":"35","author":[{"given":"Yao","family":"Chen","sequence":"first","affiliation":[{"name":"College of Pharmacy, Chongqing Medical University  Chongqing China"},{"name":"Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention (Ministry of Education) Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruichen","family":"Gao","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiajia","family":"Li","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Chongqing Medical University  Chongqing China"},{"name":"Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention (Ministry of Education) Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruijie","family":"Li","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Chongqing Medical University  Chongqing China"},{"name":"Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention (Ministry of Education) Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyu","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi","family":"Tang","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine The First Affiliated Hospital of Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xing","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Breast and Thyroid Surgery The Second Affiliated Hospital of Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1673-2826","authenticated-orcid":false,"given":"Yue","family":"Wei","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Chongqing Medical University  Chongqing China"},{"name":"Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention (Ministry of Education) Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6185-4999","authenticated-orcid":false,"given":"Linfeng","family":"Li","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Chongqing Medical University  Chongqing China"},{"name":"Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention (Ministry of Education) Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4218-9297","authenticated-orcid":false,"given":"Yuying","family":"Ma","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4370-9445","authenticated-orcid":false,"given":"Zufeng","family":"Guo","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Chongqing Medical University  Chongqing China"},{"name":"Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention (Ministry of Education) Chongqing Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,16]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11010-006-9304-6"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-032620-104421"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/mco2.70401"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41594-023-01146-w"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.20.11661"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1177319"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.5c02713"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2023.07.008"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2018.04.028"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aab0515"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2018.04.006"},{"key":"e_1_2_9_13_1","volume-title":"Irak degraders and uses thereof","author":"Mainolfi N","year":"2020"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2018.10.021"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-52966-3"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz430"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1260419"},{"key":"e_1_2_9_18_1","volume-title":"Protein degraders and uses thereof","author":"Yang B","year":"2023"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.drudis.2020.01.014"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-52061-7"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.106844.2"}],"container-title":["Protein 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